A sensor platform based on vertically aligned carbon nanofiber (CNF) electrode arrays was developed. CNF’s inherent nanometer scale, high conductivity, wide potential window, good biocompatibility and well-defined surface chemistry make them ideal candidates as biosensor electrodes. Here we present a 3x3 electrode device, with each electrode containing 40,000 carbon nanofiber nanoelectrodes. The device was demonstrated as a multiplexed immunosensor for simultaneous, label-free detection of cardiac troponin-I, C-reactive protein and myoglobin. A hydrophobic photoresist layer was used to generate wells for discretely printing antibody probes on each electrode pad. Antibodies specific to cardiac troponin-I, C-reactive protein and myoglobin were covalently bound to the CNF surface and non-specific binding is controlled through a milk protein backfill. Antibody probe immobilization and subsequent protein binding were characterized using electrochemical impedance spectroscopy and differential pulse voltammetry. Each step of the modification process resulted in changes in resistance to charge transfer due to the changes at the electrode surface upon antibody immobilization and binding to the specific cardiac protein. The real-time label free detection of the three cardiac markers from pure components and mixtures was demonstrated with high sensitivity, down to 0.2 ng/mL, and good selectivity. Detection in human blood serum did not present false positives from non-specific protein adsorption. The results demonstrate that the present sensor can serve a miniaturized, low cost device for detection of proteins in complex mixtures making this platform a good candidate for early stage diagnosis of myocardial infarction.